Therapeutic acid ceramidase compositions and methods of making and using them

ABSTRACT

The present invention relates to a therapeutic composition including a ceramidase mixture and a pharmaceutically acceptable carrier, where the ceramidase mixture includes an inactive acid ceramidase precursor and an active acid ceramidase. The invention also relates to a method of acid ceramidase treatment, including formulating the acid ceramidase used in said treatment as a ceramidase mixture, where the ceramidase mixture includes an inactive acid ceramidase precursor and an active acid ceramidase. The invention further relates to a method of producing a therapeutic composition including providing a medium containing an inactive acid ceramidase precursor; incubating the medium under conditions effective to transform a portion of the inactive acid ceramidase precursor to active acid ceramidase; and recovering the incubated medium as a ceramidase mixture comprising the inactive acid ceramidase precursor and an active acid ceramidase. The present invention also relates to preparation of a therapeutic composition of a ceramidase lacking acid sphingomyelinase.

This application is a continuation of U.S. patent application Ser. No.14/776,442, filed Mar. 13, 2014, issued as U.S. Pat. No. 9,937,246 onApr. 10, 2018, which is a national stage application under 35 U.S.C. §371 of International Patent Application No. PCT/US2014/026481, filedMar. 13, 2014, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/784,594, filed Mar. 14, 2013, which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to therapeutic acid ceramidase compositions andmethods of making and using them.

BACKGROUND OF THE INVENTION

Due to its involvement in the human genetic disorder FarberLipogranulomatosis (“FD”), Acid ceramidase (“AC;” N-acylsphingosinedeacylase, I.U.B.M.B. Enzyme No. EC 3.5.1.23) is the most extensivelystudied member of the ceramidase enzyme family. The protein has beenpurified from several sources, and the human and mouse cDNAs and geneshave been obtained (Bernardo et al., “Purification, Characterization,and Biosynthesis of Human Acid Ceramidase,” J. Biol. Chem. 270:11098-102(1995); Koch et al., “Molecular Cloning and Characterization of aFull-length Complementary DNA Encoding Human Acid Ceramidase.Identification of the First Molecular Lesion Causing Farber Disease,” J.Biol. Chem. 2711:33110-5 (1996); Li et al., “Cloning andCharacterization of the Full-length cDNA and Genomic Sequences EncodingMurine Acid Ceramidase,” Genomics 50:267-74 (1998); Li et al., “TheHuman Acid Ceramidase Gene (ASAH): Chromosomal Location, MutationAnalysis, and Expression,” Genomics 62:223-31 (1999)). Growing interestin the biology of this and other ceramidases stems from the fact thatthese enzymes play a central role in ceramide metabolism.

Ceramide is a signaling lipid that is produced in response to variousstimuli and extrinsic factors, including serum deprivation and treatmentwith many chemotherapy drugs, as well as in many human diseases (Hannun,“Function of Ceramide in Coordinating Cellular Responses to Stress,”Science 274:1855-9 (1996); Spiegel et al., “Signal Transduction ThroughLipid Second Messengers,” Curr. Opin. Cell. Biol. 8:159-67 (1996)).Inside cells, ceramide can influence growth and differentiation,regulate protein secretion, induce DNA fragmentation and apoptosis, andincrease the synthesis and secretion of cytokines. Normally present inlow amounts, in response to these factors, ceramide is rapidly producedat the cell surface, leading to membrane re-organization and downstreamsignaling that results in apoptosis. After stimulation, AC and/or otherceramidases may then hydrolyze ceramide into the individual fatty acidand sphingosine components (Gat, “Enzymic Hydrolysis and Synthesis ofCeramide,” J. Biol. Chem. 238:3131-3 (1963); Gat, “Enzymatic Hydrolysisof Sphingolipids. 1. Hydrolysis and Synthesis of Ceramides by an Enzymefrom Rat Brain,” J. Biol. Chem. 241:3724-31 (1966); Hassler & Bell,“Ceramidase: Enzymology and Metabolic Roles,” Adv. Lip. Res. 26:49-57(1993)). Because ceramide degradation is the only source ofintracellular sphingosine (Rother et al., “Biosynthesis ofSphingolipids: Dihydroceramide and Not Sphinganine Is Desaturated byCultured Cells,” Biochem. Biophys. Res. Commun. 189:14-20 (1992)), theseenzymes may also be rate-limiting steps in determining the intracellularlevels of this compound. Importantly, a derivative of sphingosine,sphingosine-1-phosphate (“S1P”), can counteract the apoptotic effects ofceramide (Cuvillier et al., “Suppression of Ceramide-mediated ProgrammedCell Death by Sphingosine-1-phosphate,” Nature 381:800-3 (1996)),leading to the suggestion that ceramidases can be “rheostats” thatmaintain a proper balance between cell growth and death (Spiegel &Merrill, “Sphingolipids Metabolism and Cell Growth Regulation,” FASEB J.10:1388-97 (1996)).

AC hydrolyzes the amide bond linking the sphingosine and fatty acidmoieties of the lipid ceramide (Park and Schuchman, “Acid Ceramidase andHuman Disease,” Biochim. Biophys. Acta. 1758(12): 2133-2138 (2006)).Ceramide, sphingosine (and its phosphorylated derivative S1P) arebioactive lipids, and thus the activity of AC must be carefullyregulated in cells (Young et al., “Sphingolipids: Regulators ofCrosstalk Between Apoptosis and Autophagy,” J. Lipid Res. 54:5-19(2013). One important mechanism by which AC activity is regulated is thecleavage of the inactive precursor polypeptide into the active enzymeconsisting of an alpha and beta subunit linked via disulfide bonds(Shtraizent et al., “Autoproteolytic Cleavage and Activation of HumanAcid Ceramidase,” J. Biol. Chem. 283:11253-11259 (2008)). It haspreviously been shown that recombinant AC produced in Chinese Hamsterovary (“CHO”) cells and secreted into the media is a mixture of inactiveprecursor and active (cleaved) enzyme (He et al., “Purification andCharacterization of Recombinant, Human Acid Ceramidase,” J. Biol. Chem.278:32978-32986 (2003)).

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a therapeuticcomposition including a ceramidase mixture and a pharmaceuticallyacceptable carrier. The ceramidase mixture includes an inactive acidceramidase precursor and an active acid ceramidase.

A second aspect of the present invention relates to a method of acidceramidase treatment, including formulating the acid ceramidase used insaid treatment as a ceramidase mixture, where the ceramidase mixtureincludes an inactive acid ceramidase precursor and an active acidceramidase.

A third aspect of the present invention relates to a method of producinga therapeutic composition. The method includes providing a mediumcontaining an inactive acid ceramidase precursor and incubating themedium under conditions effective to transform a portion of the inactiveacid ceramidase precursor to active acid ceramidase. The incubatedmedium is recovered as a ceramidase mixture comprising the inactive acidceramidase precursor and an active acid ceramidase.

The present invention describes an optimal composition of recombinant AC(rAC). The present invention further describes the novel finding that,contrary to expectation, the fully active form of the enzyme is not thebest form for promoting cell survival. Rather, preparations of purifiedrAC with higher amounts of inactive acid ceramidase (AC) precursorversus processed active AC are more effective at promoting cell survivaland/or improving cell phenotype. Two preparations of recombinant AC wereobtained containing different ratios of precursor and active enzyme.They were then used to evaluate the effects on the survival of oocytesin culture. Contrary to expectation, the preparation containing a higherratio of inactive precursor had a greater effect on cell survival. It ishypothesized that this is due to the fact that the fully active enzymehas a shorter half-life in cells and in cell culture media. The same twopreparations were tested using cultured primary chondrocytes. As withthe oocytes, the preparation of recombinant AC with less of the activeform had a greater effect on the expression of collagen 2, a marker ofchondrogenesis.

rAC is being used experimentally in a number of cell systems and animalmodels to slow ceramide-related cell death and/or improve the phenotypeof cells used for cell transplantation. It is also being studied inseveral disease models. The present invention describes the optimalpreparation of rAC to be used for these purposes, which has numerouspotential practical implications (e.g., in vitro fertilization,cartilage repair, and cystic fibrosis treatment).

In another derivative of the present invention, a novel method for thepurification of recombinant AC was developed. In this method heatinactivation was used to remove acid sphingomyelinase and othercontaminating proteins from the recombinant AC preparations. Previouswork has shown that acid sphingomyelinase, a related lipid hydrolase,tightly binds to AC and co-purifies with it (Bernardo et al.,“Purification, Characterization, and Biosynthesis of Human AcidCeramidase,” J. Biol. Chem. 270:11098-11102 (1995), which is herebyincorporated by reference in its entirety). It has now been found thatunlike most proteins, AC activity is fully stable when heated at 60° C.Thus, after column chromatography heat inactivation can be used toremove acid sphingomyelinase activity from the recombinant ACpreparation.

Together, these two novel findings regarding (i) the importance ofmaintaining an optimal ratio of precursor and active AC, and (ii) theuse of heat inactivation to remove acid sphingomyelinase activity andother contaminating proteins from the preparation, constitute unique andimportant observations regarding the composition of recombinant AC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show that preparations of rAC with less active ceramidaseperform better than those with more active ceramidase. FIG. 1A is awestern blot analysis showing the relative amounts of active(alpha/beta) versus inactive precursor rAC in two different bioreactorruns (Lot 6 and Lot 7). FIG. 1B summarizes results showing the abilityof Lots 6 and 7 to form healthy mouse embryos. FIG. 1C depicts resultsof Lot 6 and Lot 7 after testing using cultured rat chondrocytes. At twoweeks, the amount of collagen 2 expression was analyzed using westernblotting.

FIG. 2 illustrates a time-response curve of AC and acid sphingomyelinaseactivity in Lot 7. Acid sphingomyelinase activity was removed withoutaffecting AC activity.

FIG. 3 is a plot of acid ceramidase activity in (nmol/ml/hour) versusincubation time (in days).

FIG. 4 is a Western blot showing conversion of inactive acid ceramidaseto active acid ceramidase.

FIGS. 5A-B are a Western blot showing conversion of inactive acidceramidase to active acid ceramidase.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a therapeuticcomposition including a ceramidase mixture and a pharmaceuticallyacceptable carrier. The ceramidase mixture includes an inactive ACprecursor and an active AC.

Ceramidases hydrolyze the amide linkage of ceramides to generate freefatty acids and sphingoid bases (Nikolova-Karakashian et al.,“Ceramidases,” Methods Enzymol. 311:194-201 (2000); Hassler et al.,“Ceramidases: Enzymology and Metabolic Roles,” Adv. Lipid Res. 26:49-57(1993), which are hereby incorporated by reference in their entirety).There are three types of ceramidases described to date(Nikolova-Karakashian et al., “Ceramidases,” Methods Enzymol.311:194-201 (2000), which is hereby incorporated by reference in itsentirety). These are classified as acid, neutral, and alkalineceramidases according to their pH optimum of enzymatic activity.

ACs have optimal enzymatic activity at a pH<5. The human AC was thefirst ceramidase to be cloned (Koch et al., “Molecular Cloning andCharacterization of a Full-Length Complementary DNA Encoding Human AcidCeramidase. Identification Of the First Molecular Lesion Causing FarberDisease,” J. Biol. Chem. 271:33110-33115 (1996), which is herebyincorporated by reference in its entirety). It is localized in thelysosome and is mainly responsible for the catabolism of ceramide.Dysfunction of this enzyme because of a genetic defect leads to asphingolipidosis disease called Lipogranulomatosis or Farber disease(Koch et al., “Molecular Cloning and Characterization of a Full-LengthComplementary DNA Encoding Human Acid Ceramidase. Identification Of theFirst Molecular Lesion Causing Farber Disease,” J. Biol. Chem.271:33110-33115 (1996), Young et al., “Sphingolipids: Regulators ofCrosstalk Between Apoptosis and Autophagy,” J. Lipid. Res. 54:5-19(2013), which is hereby incorporated by reference in its entirety).

Inactive AC precursors and active ACs suitable for use in the ceramidasemixtures of this and all aspects of the present invention can behomologous (i.e., derived from the same species) or heterologous (i.e.,derived from a different species) to the tissue, cells, and/or subjectbeing treated. Ceramidase (e.g., AC) precursor proteins undergoautoproteolytic cleavage into the active form (composed of α- andβ-subunits). The mechanism of human AC cleavage and activation isreported in Shtraizent et al., “Autoproteolytic Cleavage and Activationof Human Acid Ceramidase,” J. Biol. Chem. 283(17):11253-11259 (2008),which is hereby incorporated by reference in its entirety). This ispromoted by the intracellular environment, and, based on highlyconserved sequences at the cleavage site of ceramidase precursorproteins across species, is expected to occur in most, if not all, celltypes. Thus, ceramidase as used herein includes both active ceramidasesand ceramidase precursor proteins, where ceramidase precursor proteinsare converted into active ceramidase proteins through autoproteolyticcleavage. Embodiments in which the precursor protein is taken up by thecell of interest and converted into active ceramidase thereby, as wellas embodiments in which the precursor protein is converted into activeceramidase by a different cell or agent (present, for example, in aculture medium), are both contemplated.

AC (N-acylsphingosine deacylase, I.U.B.M.B. Enzyme No. EC 3.5.1.23)protein has been purified from several sources, and the human and mousecDNAs and genes have been obtained. See Bernardo et al., “Purification,Characterization, and Biosynthesis of Human Acid Ceramidase,” J. Biol.Chem. 270:11098-102 (1995); Koch et al., “Molecular Cloning andCharacterization of a Full-length Complementary DNA Encoding Human AcidCeramidase. Identification of the First Molecular Lesion Causing FarberDisease,” J. Biol. Chem. 2711:33110-5 (1996); Li et al., “Cloning andCharacterization of the Full-length cDNA and Genomic Sequences EncodingMurine Acid Ceramidase,” Genomics 50:267-74 (1998); Li et al., “TheHuman Acid Ceramidase Gene (ASAH): Chromosomal Location, MutationAnalysis, and Expression,” Genomics 62:223-31 (1999), all of which arehereby incorporated by reference in their entirety. It is producedthrough cleavage of the AC precursor protein (see Ferlinz et al., “HumanAcid Ceramidase: Processing, Glycosylation, and Lysosomal Targeting,” J.Biol. Chem. 276(38):35352-60 (2001), which is hereby incorporated byreference in its entirety), which is the product of the Asah1 gene (NCBIUniGene GeneID No. 427, which is hereby incorporated by reference in itsentirety). AC protein [Homo sapien] (Accession No. AAC50907) is shownbelow in SEQ ID NO: 1.

(SEQ ID NO: 1)   1mpgrscvalv llaaayscav aqhappwted crkstyppsg ptyrgavpwy tinldlppyk  61rwhelmldka pmlkvivnsl knmintfvps gkvmqvvdek lpgllgnfpg pfeeemkgia 121avtdiplgei isfnifyelf ti ctsivaed kkghlihgrn mdfgvflgwn inndtwvite 181qlkpltvnld fqrnnktvfk assfagyvgm ltgfkpglfs ltlnerfsin ggylgilewi 241lgkkdamwig fltrtvlens tsyeeaknll tktkilapay filggnqsge gcvitrdrke 301sldvyeldak qgrwyvvqtn ydrwkhpffl ddrrtpakmc lnrtsqenis fetmydvlst 361kpvlnkltvy ttlidvtkgq fetylrdcpd pcigwThe AC alpha subunit begins at the amino acid at position 22 andcontinues through position 142 (as shown in bold in SEQ ID NO: 1), whilethe beta subunit of the AC begins with the amino acid at position 143and continues through position 395 (as shown in italics in SEQ ID NO:1).

Active ACs and inactive AC precursor proteins that can be used in thisand all aspects of the present invention include, without limitation,those set forth in Table 1 below.

TABLE 1 Exemplary Acid Ceramidase Family Members Homo sapiens (SEQ IDNO: 1) UniProt Q13510, Q9H715, Q96AS2 OMIM 228000 NCBI Gene 427 NCBIRefSeq NP_808592, NP_004306 NCBI RefSeq NM_177924, NM_004315 NCBIUniGene 427 NCBI Accession Q1351O, AAC73009, AAC50907 Mus musculus (SEQID NO: 2) UniProt Q9WV54, Q3U8A7, NCBI Gene 11886 NCBI RefSeq NP_062708NCBI RefSeq NM_019734 NCBI UniGene 11886 NCBI Accession AK1512O8,AK034204 Gallus gallus (SEQ ID NO: 3) UniProt Q5ZK58 NCBI Gene 422727NCBI RefSeq NP_001006453 NCBI RefSeq NM_001006453 NCBI UniGene 422727NCBI Accession CAG31885, AJ720226 Pan troglodytes (SEQ ID NO: 4) NCBIGene 464022 NCBI RefSeq XP_519629 NCBI RefSeq XM_519629 NCBI UniGene464022 Caenorhabditis elegans (SEQ ID NO: 5) UniProt O45686 IntActO45686 NCBI Gene 173120 NCBI RefSeq NP_493173 NCBI RefSeq NM_060772 NCBIUniGene 173120 NCBI Accession O45686, CAB05556 Danio rerio (SEQ ID NO:6) UniProt Q5XJR7 NCBI Gene 450068 NCBI RefSeq NP_001006088 NCBI RefSeqNM_001006088 NCBI UniGene 450068 NCBI Accession AAH83231, CB360968Rattus norvegicus (SEQ ID NO: 7) UniProt Q6P7S1, Q9EQJ6 NCBI Gene 84431NCBI RefSeq NP_445859 NCBI RefSeq NM_053407 NCBI UniGene 84431 NCBIAccession AAH61540, AF214647

The ceramidase mixture of the therapeutic composition may, in someembodiments, contain a greater amount of the inactive AC precursor thanactive AC. Alternatively, the ceramidase mixture of the therapeuticcomposition may, in some instances, contain a lesser amount of inactiveAC precursor than active AC.

In some embodiments, an effective amount of the inactive AC precursorcompared to the active AC in the mixture ranges from about 5 to 95 wt %of the inactive AC precursor and 95 to 5 wt % of the active AC; 20 to 80wt % of the inactive AC precursor and 80 to 20 wt % of the active AC; 30to 70 wt % of the inactive AC precursor and 70 to 30 wt % of the activeAC; 40 to 60 wt % of the inactive AC precursor and 60 to 40 wt % of theactive AC; 55 to 95 wt % of the inactive AC precursor and 45 to 5 wt %of the active AC; 70 to 95 wt % of the inactive AC precursor and 30 to 5wt % of the active AC; and may alternatively range from 80 to 90 wt % ofthe inactive AC precursor and 20 to 10 wt % of the active AC. Aneffective amount of the inactive AC precursor is 90 wt % while theactive ceramidase is 10 wt % of the mixture. An alternative embodimentmay include 80 wt % of the inactive ceramidase precursor and 20 wt % ofthe active AC in the ceramidase mixture. In yet a further embodiment,the ceramidase mixture may contain 60 wt % inactive ceramidase precursorand 40 wt % active ceramidase.

The therapeutic composition may also include pharmaceutically acceptableadjuvants, excipients, and/or stabilizers, and can be in solid or liquidform, such as tablets, capsules, powders, solutions, suspensions, oremulsions. Suitable adjuvants include, but are not limited to,flagellin, Freund's complete or incomplete adjuvant, aluminum hydroxide,lysolecithin, pluronic polyols, polyanions, peptides, oil emulsion,dinitrophenol, iscomatrix, and liposome polycation DNA particles.

A second aspect of the present invention relates to a method of ACtreatment, including formulating the AC used in said treatment as aceramidase mixture, where the ceramidase mixture includes an inactive ACprecursor and an active AC.

Treatment according to this aspect of the present invention is carriedout using methods that will be apparent to the skilled artisan. For adiscussion of AC in the context of human disease, see Park et al., “AcidCeramidase and Human Disease,” Biochim. Phiophys. Act. 1758:2133-2138(2006) and Zeidan et al., “Molecular Targeting of Acid Ceramidase:Implications to Cancer Therapy,” Curr. Drug Targets 9(8):653-661 (2008),both of which are hereby incorporated by reference in their entirety).

In some embodiments, treatment is carried out by introducing aceramidase protein into the cells. An approach for delivery of proteinsor polypeptide agents (e.g., active ceramidase, inactive ceramidaseprecursor proteins) involves the conjugation of the desired protein orpolypeptide to a polymer that is stabilized to avoid enzymaticdegradation of the conjugated protein or polypeptide. Conjugatedproteins or polypeptides of this type are described in U.S. Pat. No.5,681,811 to Ekwuribe, which is hereby incorporated by reference in itsentirety.

Yet another approach for delivery of proteins or polypeptide agentsinvolves preparation of chimeric proteins according to U.S. Pat. No.5,817,789 to Heartlein et al., which is hereby incorporated by referencein its entirety. The chimeric protein can include a ligand domain andthe polypeptide agent (e.g., rAC, active AC, other ceramidase, inactiveAC precursor protein, other ceramidase precursor proteins). The liganddomain is specific for receptors located on a target cell. Thus, whenthe chimeric protein is delivered to the cell, the chimeric protein willadsorb to the targeted cell, and the targeted cell will internalize thechimeric protein.

Further embodiments of the present aspect relate to methods of treatmentfor a certain disease or disorder. These methods involve formulating theAC used in the treatment as a ceramidase mixture including an inactiveceramidase precursor and an active AC.

In one embodiment, the disease or disorder is a joint disease ordisorder and the ceramidase mixture according to the methods of thepresent invention is administered to a subject to treat the subject forthe joint disease or disorder. Exemplary types of joint disease ordisorders include, without limitation, osteoarthritis, rheumatoidarthritis, mucopolysaccharidosis, degenerative joint disease, jointinjury, and Farber lipogranulomatosis.

In another embodiment, the disease or disorder is a neurodegenerativedisease or disorder and the ceramidase mixture according to the methodsof the present invention is administered to a subject to treat thesubject for the neurodegenerative disease or disorder. Exemplary typesof neurodegenerative diseases or disorders include, without limitation,Alzheimer's disease, Frontotemporal Dementia, Dementia with Lewy Bodies,Prion disease, Parkinson's disease, Huntington's disease, ProgressiveSupranuclear Palsy, Corticobasal Degeneration, Multiple System Atrophy,amyotrophic lateral sclerosis, inclusion body myositis, degenerativemyopathy, spinocerebellar atrophy, metabolic neuropathy, diabeticneuropathy, endocrine neuropathy, orthostatic hypotension, brain injury,spinal cord injury, stroke, and motor neuron diseases such as spinalmuscular atrophy.

In another embodiment, the disease or disorder is a cardiac disease ordisorder and the ceramidase mixture according to the methods of thepresent invention is administered to a subject to treat the subject forthe cardiac disease or disorder. Exemplary types of cardiac diseases ordisorders include, without limitation, heart disease, cardiac injury,atherosclerosis, thrombosis, cardiomyocyte apoptosis, hypercardia, heartinfarction, mitral regurgitation, aortic regurgitation, septal defect,and tachycardia-bradycardia syndrome.

In another embodiment, the disease or disorder is diabetes and theceramidase mixture according to the methods of the present invention isadministered to a subject to treat the subject for diabetes.

In another embodiment, the disease or disorder is a pathogenic infectionin a subject having cystic fibrosis, chronic obstructive pulmonarydisease (COPD), and/or an open wound, and the ceramidase mixtureaccording to the methods of the present invention is administered to asubject to treat the subject for the pathogenic infection. Exemplarytypes of pathogenic infections include, without limitation, viral,fungal, prionic, and bacterial.

Subjects suffering from cystic fibrosis, COPD, and/or an open wound, maypossess a high susceptibility for acquiring acute and/or chronicpathogenic infections, such as, e.g., bacterial, viral, fungal,protozoan, and/or prionic pathogenic infections. Bacterial pathogensinclude, without limitation, Bacillus anthraces, Bordetella pertussis,Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis,Clostridium botulinum, Clostridium tetani, Corynebacterium dipththeriae,Escherichia coli, enterohemorrhagic E. coli, enterotoxigenic E. coli,Haemophilus influenzae type B and non-typable, Helicobacter pylori,Legionella pneumophila, Listeria monocytogenes, Mycobacterium spp.,Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus spp., Pseudomonas aeruginosa,Rickettsia, Salmonella spp., Shigella spp., Staphylococcus spp.,Staphylococcus aureus, Streptococcus spp., Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus B, Group A beta hemolyticStreptococcus, Streptococcus mutans, Treponema pallidum, Vibriocholerae, and Yersinia pestis. In some embodiments, the pathogenicinfection is a Pseudomonas infection.

Viral pathogens include, without limitation, RNA viruses, DNA viruses,adenovirdiae (e.g., mastadenovirus and aviadeno virus), herpesviridae(e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplexvirus 5, and herpes simplex virus 6), leviviridae (e.g., levivirus,enterobacteria phage MS2, allolevirus), poxyiridae (e.g.,chordopoxyirinae, parapoxvirus, avipoxvirus, capripoxvirus,leporipoxvirus, suipoxvirus, molluscipox virus, and entomopoxyirinae),papovaviridae (e.g., polyomavirus and papillomavirus), paramyxoviridae(e.g., paramyxovirus, parainfluenza virus 1, mobillivirus such asmeasles virus, rubulavirus (such as mumps virus), pneumonoviridae (e.g.,pneumovirus, human respiratory syncytial virus), metapneumovirus (e.g.,avian pneumovirus and human metapneumo virus), picomaviridae (e.g.,enterovirus, rhinovirus, hepatovirus such as human hepatitis A virus,cardiovirus, and apthovirus), reoviridae (e.g., orthoreo virus,orbivirus, rotavirus, cypo virus, fijivirus, phytoreo virus, andoryzavirus), retroviridae (e.g., mammalian type B retroviruses,mammalian type C retroviruses, avian type C retroviruses, type Dretrovirus group, BLV-HTLV retroviruses, lentivirus (such as humanimmunodeficiency virus 1 and human immunodeficiency virus 2; and spumavirus), flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g.,hepatitis B virus), togaviridae (e.g., alphavirus—such as sindbis virusand rubivirus, such as rubella virus), rhabdoviridae (e.g.,vesiculovirus, lyssavirus, ephemera virus, cytorhabdovirus, andnecleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocyticchoriomeningitis virus, Ippy virus, and lassa virus), and coronaviridae(e.g., coronavirus and torovirus), Cytomegalovirus (mononucleosis),Dengue virus (dengue fever, shock syndrome), Epstein-Barr virus(mononucleosis, Burkitt's lymphoma), Human T-cell lymphotropic virustype 1 (T-cell leukemia), Influenza A, B, and C (respiratory disease),Japanese encephalitis virus (pneumonia, encephalopathy), Poliovirus(paralysis), Rhinovirus (common cold), Rubella virus (fetalmalformations), Vaccinia virus (generalized infection), Yellow fevervirus (jaundice, renal and hepatic failure), and Varicella zoster virus(chickenpox).

Pathogenic fungi include, without limitation, the genera Aspergillus(e.g., Aspergillus fumigates), Blastomyces, Candida (e.g., Candidaalbicans), Coccidiodes, Cryptococcus, Histoplasma, Phycomyces, Tineacorporis, Tinea unguis, Sporothrix schenckii, and Pneumocystis carinii.Pathogenic protozoan include, without limitation, Trypanosome spp.,Leishmania spp., Plasmodium spp., Entamoeba spp., and Giardia spp. suchas Giardia lamblia.

As described herein, an “open wound” refers to a type of injury in whichan epithelial layer, i.e., skin, is torn, cut, and/or punctured. In someembodiments, an open wound refers to a sharp injury which damages thedermis of the skin and concomitantly increases the chance of acquiringan infection. The term “open wound” also encompasses burns.

In another embodiment, the disease or disorder is an infection caused byceramide accumulation and the ceramidase mixture according to themethods of the present invention is administered to a subject to treatthe subject for the ceramide accumulation infection.

The present invention may, in other embodiments, be used to treat Farberdisease.

In at least one embodiment, treatment is carried out in vitro. In thisembodiment, a ceramidase mixture can be taken from the subject or from asecond subject then administered to the first subject (e.g., byinjecting the mixture into the first subject). In at least oneembodiment, treatment is carried out in vivo.

Mammalian subjects according to these aspects of the present inventioninclude, for example, human subjects, equine subjects, porcine subjects,feline subjects, and canine subjects. Human subjects are particularlypreferred.

In all embodiments that involve administering the ceramidase mixture toa subject, any combination of active ceramidase, ceramidase precursorprotein, and/or nucleic acid encoding ceramidase/ceramidase precursorprotein can be administered. Administration can be accomplished eithervia systemic administration to the subject or via targetedadministration to affected tissues, organs, and/or cells. The ceramidasemixture may be administered to a non-targeted area along with one ormore agents that facilitate migration of the ceramidase mixture to(and/or uptake by) a targeted tissue, organ, or cell. Additionallyand/or alternatively, the ceramidase mixture itself can be modified tofacilitate its transport to (and uptake by) the desired tissue, organ,or cell, as will be apparent to one of ordinary skill in the art.

Typically, the ceramidase mixture will be administered to a subject in avehicle that delivers the ceramidase to the target cell, tissue, ororgan. Exemplary routes of administration include, without limitation,orally, by inhalation, intratracheal inoculation, aspiration, airwayinstillation, aerosolization, nebulization, intranasal instillation,oral or nasogastric instillation, intraperitoneal injection,intravascular injection, topically, transdermally, parenterally,subcutaneously, intravenous injection, intra-arterial injection (such asvia the pulmonary artery), intramuscular injection, intrapleuralinstillation, intraventricularly, intralesionally, intrathecally, byapplication to mucous membranes (such as that of the nose, throat,bronchial tubes, genitals, and/or anus), or implantation of a sustainedrelease vehicle.

In some embodiments, the ceramidase mixture is administered orally,topically, intranasally, intraperitoneally, intravenously,subcutaneously, or by aerosol inhalation. In some embodiments, theceramidase mixture is administered via aerosol inhalation. In someembodiments, the ceramidase mixture can be incorporated intopharmaceutical compositions suitable for administration, as describedherein.

The ceramidase mixture may be orally administered, for example, with aninert diluent, or with an assimilable edible carrier, or they may beenclosed in hard or soft shell capsules, or they may be compressed intotablets, or may be incorporated directly with the food of the diet. Fororal therapeutic administration, the ceramidase mixture may beincorporated with excipients and used in the form of tablets, capsules,elixirs, suspensions, syrups, and the like. Such compositions andpreparations should contain at least 0.1% of ceramidase. The percentageof ceramidase mixture in these compositions may, of course, be variedand may conveniently be between about 2% to about 60% of the weight ofthe unit. The amount of the ceramidase mixture in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, or alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as fatty oil.

The ceramidase mixture may also be administered parenterally. Solutionsor suspensions of ceramidase can be prepared in water suitably mixedwith a surfactant, such as hydroxypropylcellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof in oils. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solutions, and glycols such as propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The ceramidase mixture may also be administered directly to the airwaysin the form of an aerosol. For use as aerosols, ceramidase in solutionor suspension may be packaged in a pressurized aerosol containertogether with suitable propellants, for example, hydrocarbon propellantslike propane, butane, or isobutane with conventional adjuvants. Theceramidase mixture may also be administered in a non-pressurized form.

Exemplary delivery devices include, without limitation, nebulizers,atomizers, liposomes (including both active and passive drug deliverytechniques) (Wang et al., “pH-Sensitive Immunoliposomes MediateTarget-cell-specific Delivery and Controlled Expression of a ForeignGene in Mouse,” Proc. Nat'l Acad. Sci. USA 84:7851-5 (1987); Bangham etal., “Diffusion of Univalent Ions Across the Lamellae of SwollenPhospholipids,” J. Mol. Biol. 13:238-52 (1965); U.S. Pat. No. 5,653,996to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau et al.; and U.S. Pat.No. 5,059,421 to Loughrey et al.; Wolff et al., “The Use of MonoclonalAnti-Thy1 IgG1 for the Targeting of Liposomes to AKR-A Cells in Vitroand in Vivo,” Biochim. Biophys. Acta 802:259-73 (1984), each of which ishereby incorporated by reference in its entirety), transdermal patches,implants, implantable or injectable protein depot compositions, andsyringes. Other delivery systems which are known to those of skill inthe art can also be employed to achieve the desired delivery ofceramidase to the desired organ, tissue, or cells.

Administration can be carried out as frequently as required and for aduration that is suitable to provide effective treatment. For example,administration can be carried out with a single sustained-release dosageformulation or with multiple daily doses.

Treatment according to this and all aspects of the present invention maybe carried out in vitro or in vivo. In vivo treatments include, forexample, embodiments in which the population of cells is present in amammalian subject. In such embodiments the population of cells can beeither autologous (produced by the subject), homologous, orheterologous. Suitable subjects according to these embodiments includemammals, e.g., human subjects, equine subjects, porcine subjects, felinesubjects, and canine subjects.

In one embodiment, one or more additional agents which reduce ceramidelevels may be administered with the ceramidase mixture. This includes,without limitation, inhibitors of acid sphingomyelinase (e.g.,amitryptyline (Becker et al., “Acid Sphingomyelinase InhibitorsNormalize Pulmonary Ceramide and Inflammation in Cystic Fibrosis,” Am.J. Respir. Cell. Mol. Biol. 42:716-724 (2010), which is herebyincorporated by reference in its entirety) and inhibitors of ceramidesynthases (e.g., Shiffmann et al., “Inhibitors of Specific CeramideSynthases,” Biochimie 94:558-565 (2012), which is hereby incorporated byreference in its entirety)).

The effective amount of a therapeutic agent/cell population of thepresent invention administered to the subject will depend on the typeand severity of the disease or disorder and on the characteristics ofthe individual, such as general health, age, sex, body weight, andtolerance to drugs. It will also depend on the degree, severity, andtype of disease or disorder. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors.

In one embodiment of the present invention, the method includes treatingone or more mammalian cells ex vivo with said ceramidase mixture topromote cell survival. Cells whose survival can be promoted according tothis aspect of the present invention include, without limitation, thosethat utilize the ceramidase apoptosis pathway, which includes a widevariety of cells (Obeid et al., “Programmed Cell Death Induced byCeramide,” Science 259:1769-71 (1993), which is hereby incorporated byreference in its entirety), e.g., hepatocytes (Arora et al., “CeramideInduces Hepatocyte Cell Death Through Disruption of MitochondrialFunction in the Rat,” Hepatol. 25:958-63 (1997), which is herebyincorporated by reference in its entirety), skin fibroblasts (Mizushimaet al., “Ceramide, a Mediator of Interleukin 1, Tumour Necrosis Factorα, as Well as Fas Receptor Signalling, Induces Apoptosis of RheumatoidArthritis Synovial Cells,” Ann. Rheum. Dis. 57:495-9 (1998), which ishereby incorporated by reference in its entirety), chondrocytes (MacRaeet al., “Ceramide Inhibition of Chondrocyte Proliferation and BoneGrowth Is IGF-I Independent,” J. Endocrinol. 191(2):369-77 (2006), whichis hereby incorporated by reference in its entirety), lung epithelium(Chan & Goldkorn, “Ceramide Path in Human Lung Cell Death,” Am. J.Respir. Cell Mol. Biol. 22(4):460-8 (2000), which is hereby incorporatedby reference in its entirety), erythrocytes (Lang et al., “Mechanisms ofSuicidal Erythrocyte Death,” Cell. Physiol. Biochem. 15:195-202 (2005),which is hereby incorporated by reference in its entirety),cardiomyocytes (Parra et al., “Changes in Mitochondrial Dynamics DuringCeramide-induced Cardiomyocyte Early Apoptosis,” Cardiovasc. Res.(2007), which is hereby incorporated by reference in its entirety), andlymphocytes (Gombos et al., “Cholesterol and Sphingolipids as LipidOrganizers of the Immune Cells' Plasma Membrane: Their Impact on theFunctions of MHC Molecules, Effector T-lymphocytes and T-cell Death,”Immunol. Lett. 104(1-2):59-69 (2006), which is hereby incorporated byreference in its entirety), eggs, embryos, neurons, sperm, synovialfibroblasts, and embryonic stem cells. Preferred cell types are eggs(fertilized or unfertilized), embryos, primary cells (e.g., neurons),sperm, synovial fibroblasts, and embryonic stem cells. Moreover, theceramide apoptosis pathway appears to be conserved across mammalianspecies (Lee & Amoscato, “TRAIL and Ceramide,” Vitam. Horm. 67:229-55(2004); see also, Samadi, “Ceramide-induced Cell Death in LensEpithelial Cells,” Mol. Vis. 13:1618-26 (2007) (humans); Parra et al.,“Changes in Mitochondrial Dynamics During Ceramide-induced CardiomyocyteEarly Apoptosis,” Cardiovasc. Res. (2007) (rat); de Castro E Paula etal., “Ceramide Inhibits Development and Cytokinesis and InducesApoptosis in Preimplantation Bovine Embryos,” Mol. Reprod. Devel., DOINo. 10.1002/mrd.20841 (2007) (cows), each which is hereby incorporatedby reference in its entirety). Therefore, it is expected that, for eachof the cell types recited above, suitable cells include those of humans,monkeys, mice, rats, guinea pigs, cows, horses, sheep, pigs, dogs, andcats. This method may also be used to prolong the survival of eggsand/or embryos during in vitro fertilization procedures, facilitatingthe identification and selection of healthy embryos for reimplantation,especially for older human women and for veterinary breeding procedures.

Cells according to this aspect of the present invention can be providedby methods that will be apparent to the skilled artisan. By way ofexample, the cells can be obtained from an animal or from an existing exvivo source (e.g., a tissue sample, a cell culture, etc.) using standardtechniques. Treating cells ex vivo includes treating cells present in ahomogeneous culture, as well as cells present in a heterogeneous culture(e.g., a tissue sample).

Inactive AC precursors and active ACs that can be used to prepare theceramidase mixture in this and all aspects of the present inventioninclude, without limitation, those set forth in Table 1, supra. In thisand all aspects of the present invention (including the in vivo methodsdiscussed below), the AC can be homologous (i.e., derived from the samespecies) or heterologous (i.e., derived from a different species) to theone or more cells being treated.

One embodiment of the present aspect of AC treatment relates to a methodof producing chondrocytes with the ceramidase mixture. This methodinvolves selecting a population of cells having the potential todifferentiate into chondrocytes and treating the selected cellpopulation with the ceramidase mixture to transform one or more of thecells in the selected population into chondrocytes.

Cells having the potential to differentiate into chondrocytes includebone marrow cells, fibroblasts, mesenchymal stem cells, and/orfibroblasts (see Mizushima et al., “Ceramide, a Mediator of Interleukin1, Tumour Necrosis Factor α, as Well as Fas Receptor Signaling, InducesApoptosis of Rheumatoid Arthritis Synovial Cells,” Ann. Rheum. Dis.57:495-9 (1998), which is hereby incorporated by reference in itsentirety).

Chondrocytes according to this aspect of the present invention include,without limitation, articular chondrocytes, nasal chondrocytes, trachealchondrocytes, meniscal chondrocytes, and aural chondrocytes. Theseinclude, for example, mammalian chondrocytes, e.g., human chondrocytes,equine chondrocytes, porcine chondrocytes, feline chondrocytes, andcanine chondrocytes. Preferably, the chondrocytes are primarychondrocytes.

Suitable cells according to this and all other aspects of the presentinvention include mammalian cells, e.g., human cells, equine cells,porcine cells, feline cells, and/or canine cells. Human cells areparticularly preferred.

In this and all aspects of the present invention involving cellpopulations, embodiments in which the cells are all of one type, as wellas embodiments in which the population is a mixture of two or more celltypes, are both contemplated.

The ceramidase mixture and methods of treating the populations of cellswith ceramidase mixture include all those set forth supra.

Another embodiment of the present aspect of AC treatment relates to amethod of promoting chondrogenesis with the ceramidase mixture. In oneembodiment, this method further includes selecting a population of stemcells in need of differentiation into chondrocytes, treating thepopulation of stem cells with the ceramidase mixture to enrichmesenchymal stem cells within the stem cell population, and treating thepopulation of enriched mesenchymal stem cells with the ceramidasemixture to promote differentiation of mesenchymal stem cells intochondrocytes.

Suitable cells populations according to this aspect of the presentinvention include mammalian cells populations, e.g., human cellspopulations, equine cells populations, porcine cells populations, felinecells populations, and/or canine cells populations. Human cellspopulations are particularly preferred.

Suitable stem cells according to this and all other aspects of thepresent invention include bone marrow cells, adipocytes, and skin cells.Additional stem cells according to this aspect of the present inventioninclude, without limitation, embryonic stem cells, somatic stem cells,induced pluripotent stem cells, totipotent stem cells, pluripotent stemcells, and multipotent stem cells. Exemplary stem cells include, forexample, hematopoietic stem cells, mesenchymal stem cells, neural stemcells, endothelial progenitor cells, epithelial stem cells, epidermalstem cells, adipocytes, and cardiac stem cells. Suitable stem cellsinclude, but are not limited to, mammalian cells, e.g., human, equine,porcine, feline, and canine bone marrow cells, adipocytes, and skincells. Human cells are particularly preferred.

Suitable chondrocytes are consistent with those described supra. Thedifferentiated mesenchymal stem cells may, alternatively, be primarycells such as, but not limited to, neurons, hepatocytes, bone cells,lung cells, and cardiac cells.

In at least one embodiment, the number of differentiated cells in thecell population is maintained. In at least one embodiment, the number ofdifferentiated cells in the cell population is increased. As will beapparent to the skilled artisan, maintaining or increasing the overallnumber of differentiated cells in the population can be achieved bydecreasing or preventing de-differentiation of cells in the populationthat are already differentiated, by stimulating the differentiation ofundifferentiated cells in the population, or both.

The ceramidase mixture and methods of treating the populations of cellswith ceramidase mixture include all those set forth supra.

A third aspect of the present invention relates to a method of producinga therapeutic composition. The method includes providing a mediumcontaining an inactive AC precursor; incubating the medium underconditions effective to transform a portion of the inactive AC precursorto active AC; and recovering the incubated medium as a ceramidasemixture comprising the inactive AC precursor and an active AC.

The therapeutic composition of the present invention contains arecombinant protein including both inactive AC precursor and active AC.The recombinant protein of the present invention may be prepared for usein the above described methods of the present invention using standardmethods of synthesis known in the art, including solid phase peptidesynthesis (Fmoc or Boc strategies) or solution phase peptide synthesis.Alternatively, proteins of the present invention may be prepared usingrecombinant expression systems.

Generally, the use of recombinant expression systems involves insertingthe nucleic acid molecule encoding the amino acid sequence of thedesired peptide into an expression system to which the molecule isheterologous (i.e., not normally present). One or more desired nucleicacid molecules encoding a peptide of the invention may be inserted intothe vector. When multiple nucleic acid molecules are inserted, themultiple nucleic acid molecules may encode the same or differentpeptides. The heterologous nucleic acid molecule is inserted into theexpression system or vector in proper sense (5′→3′) orientation relativeto the promoter and any other 5′ regulatory molecules, and correctreading frame.

The preparation of the nucleic acid constructs can be carried out usingstandard cloning procedures well known in the art as described by JosephSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold SpringsHarbor 1989). U.S. Pat. No. 4,237,224 to Cohen and Boyer, which ishereby incorporated by reference in its entirety, describes theproduction of expression systems in the form of recombinant plasmidsusing restriction enzyme cleavage and ligation with DNA ligase. Theserecombinant plasmids are then introduced by means of transformation intoa suitable host cell.

A variety of genetic signals and processing events that control manylevels of gene expression (e.g., DNA transcription and messenger RNA(“mRNA”) translation) can be incorporated into the nucleic acidconstruct to maximize peptide production. For the purposes of expressinga cloned nucleic acid sequence encoding a desired recombinant protein,it is advantageous to use strong promoters to obtain a high level oftranscription. Depending upon the host system utilized, any one of anumber of suitable promoters may be used. For instance, when cloning inE. coli, its bacteriophages, or plasmids, promoters such as the T7 phagepromoter, lac promoter, trp promoter, recA promoter, ribosomal RNApromoter, the P_(R) and P_(L) promoters of coliphage lambda and others,including but not limited, to lacUV5, ompF, bla, lpp, and the like, maybe used to direct high levels of transcription of adjacent DNA segments.Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. colipromoters produced by recombinant DNA or other synthetic DNA techniquesmay be used to provide for transcription of the inserted gene. Commonpromoters suitable for directing expression in mammalian cells include,without limitation, SV40, MMTV, metallothionein-1, adenovirus Ela, CMV,immediate early, immunoglobulin heavy chain promoter and enhancer, andRSV-LTR. Mammalian cells that may be used for manufacture of therecombinant protein of the present invention include, for example,Chinese Hamster Ovary (CHO) cells, plant cells, chicken eggs, and humanfibroblasts.

There are other specific initiation signals required for efficient genetranscription and translation in prokaryotic cells that can be includedin the nucleic acid construct to maximize peptide production. Dependingon the vector system and host utilized, any number of suitabletranscription and/or translation elements, including constitutive,inducible, and repressible promoters, as well as minimal 5′ promoterelements, enhancers or leader sequences may be used. For a review onmaximizing gene expression see Roberts and Lauer, “Maximizing GeneExpression On a Plasmid Using Recombination In Vitro,” Methods inEnzymology 68:473-82 (1979), which is hereby incorporated by referencein its entirety.

A nucleic acid molecule encoding a recombinant protein of the presentinvention, a promoter molecule of choice, including, without limitation,enhancers, and leader sequences; a suitable 3′ regulatory region toallow transcription in the host, and any additional desired components,such as reporter or marker genes, are cloned into the vector of choiceusing standard cloning procedures in the art, such as described inJoseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (ColdSprings Harbor 1989); Frederick M. Ausubel, SHORT PROTOCOLS IN MOLECULARBIOLOGY (Wiley 1999), and U.S. Pat. No. 4,237,224 to Cohen and Boyer,which are hereby incorporated by reference in their entirety.

Once the nucleic acid molecule encoding the peptide has been cloned intoan expression vector, it is ready to be incorporated into a host.Recombinant molecules can be introduced into cells, without limitation,via transfection (if the host is a eukaryote), transduction,conjugation, mobilization, or electroporation, lipofection, protoplastfusion, mobilization, or particle bombardment, using standard cloningprocedures known in the art, as described by JOSEPH SAMBROOK et al.,MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989), whichis hereby incorporated by reference in its entirety.

A variety of suitable host-vector systems may be utilized to express therecombinant protein or polypeptide. Primarily, the vector system must becompatible with the host used. Host-vector systems include, withoutlimitation, the following: bacteria transformed with bacteriophage DNA,plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria.

Purified peptides may be obtained by several methods readily known inthe art, including ion exchange chromatography, hydrophobic interactionchromatography, affinity chromatography, gel filtration, and reversephase chromatography. The peptide is preferably produced in purifiedform (preferably at least about 80% or 85% pure, more preferably atleast about 90% or 95% pure) by conventional techniques. Depending onwhether the recombinant host cell is made to secrete the peptide intogrowth medium (see U.S. Pat. No. 6,596,509 to Bauer et al., which ishereby incorporated by reference in its entirety), the peptide can beisolated and purified by centrifugation (to separate cellular componentsfrom supernatant containing the secreted peptide) followed by sequentialammonium sulfate precipitation of the supernatant. In one embodiment ofthe present invention, cells may be transformed with DNA encoding AC andthen cultured under conditions effective to produce the mediumcontaining inactive AC precursor. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by otherchromatography.

In one embodiment of the present invention, the incubation is carriedout under conditions effective to reduce the transformation rate ofinactive AC precursor to active AC compared to the transformation rateachieved when said incubating is carried out at a pH of 4 and atemperature of 4° C. or 37° C., for 24 hours, under otherwise consistentconditions. Alternatively, the incubating may be carried out underconditions effective to enhance the transformation rate of inactive ACprecursor to active AC compared to those same conditions.

In some embodiments, the ceramidase mixture during the incubating mayhave a pH over 4.0 and up to 6.5. The mixture may, for example, have apH of 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5. In other embodiments, thetemperature of the ceramidase mixture during said incubating may be atleast −30° C. and under 37° C. The temperature of the mixture may, forexample, be −30° C., −25° C., −20° C., −15° C., −10° C., −5° C., 0° C.,5° C., 10° C., 15° C., 20° C., 25° C., 30° C., or 35° C. Alternatively,the mixture may be incubated under conditions of −30° C. with a pH of4.0, 4° C. with a pH of 4.0 or 6.5, 25° C. with a pH of 4.0, or 37° C.with a pH of 4.0. The mixture may be incubated for a period of time suchas, but not limited to, approximately 30 minutes, 1 hour, 3 hours, 30hours, or 300 hours.

During incubation of this aspect of the present invention, the mediummay be heated under conditions effective to remove acid sphingomyelinaseactivity. In this embodiment, the medium may be heated to 60° C. for aperiod of time including, but not limited to, less than 20 minutes,20-40 minutes, 40-60 minutes, or more than 60 minutes.

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the present invention are described invarious levels of detail in order to provide a substantial understandingof the present technology. The definitions of certain terms as used inthis specification are also provided. Unless defined otherwise, alltechnical and scientific terms used herein generally have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1—Materials and Methods

Preparation of rAC (Lot 6 and Lot 7)—

Chinese hamster ovary cells overexpressing the human Asah1 gene weregenerated and rAC was purified from the media (He et al., “Purificationand Characterization of Recombinant, Human Acid Ceramidase. CatalyticReactions and Interactions With Acid Sphingomyelinase,” J. Biol. Chem.278:32978-32986 (2003), which is hereby incorporated by reference in itsentirety). No in vitro manipulation was carried out after purificationof Lot 6 (higher amount of inactive AC precursor). After purification ofLot 7 enzyme, the rAC was incubated in pH 4 citrate phosphate buffer at37° C. for three hours.

Comparison of Lot 6 and Lot 7 for Mouse Embryo Production—

Methods for using rAC for mouse embryo production are described inEliyahu et al., “Acid Ceramidase Improves the Quality of Oocytes andEmbryos and the Outcome of In Vitro Fertilization,” FASEB J.24:1229-1238 (2010), which is hereby incorporated by reference in itsentirety. Sperm and mature oocytes were obtained from C57 Black mice andin vitro fertilization was carried out using equal amounts of Lot 6 andLot 7 enzyme.

Comparison of Lot 6 and Lot 7 to Improve the Chondrogenic Phenotype ofRat Articular Chondrocytes—

Equal amounts of Lot 6 and Lot 7 rAC were added to the media of primaryarticular chondrocytes isolated from femurs. Cartilage was digested andcells were placed into culture with and without rAC supplementation.After three days the media was changed to media without rAC. Cells weregrown for an additional two weeks and the levels of collagen 2 (markerof mature articular chondrocytes) was determined by western blotting.

Example 2—rAC with Less Active AC Perform Better than Those with MoreActive AC

Preparations of rAC with less active ceramidase perform better thanthose with more active ceramidase (FIGS. 1A-1C). As indicated in FIG.1A, a western blot analysis showed the relative amounts of active(alpha/beta) versus inactive precursor rAC in two different bioreactorruns (Lot 6 and Lot 7). Lot 7 had more active rAC than in Lot 6. Invitro examples (IVF and chondrocytes) compared two rAC preparations withratios that were approximately 90:10 (inactive:active) (Lot 6) versusapproximately 80:20 (inactive:active) (Lot 7) (FIG. 1A). Apoptosis wasdetermined at 24 hours using standard morphological methods (e.g.,membrane integrity, etc.) (Eliyahu et al., “Acid Ceramidase Improves theQuality of Oocytes and Embryos and the Outcome of In VitroFertilization,” FASEB J. 24:1229-1238 (2010), which is herebyincorporated by reference in its entirety).

FIG. 1B summarizes results for ability to form healthy mouse embryos inLot 6 and Lot 7. Lot 7 (containing more active rAC) produced moreapoptotic embryos than lot 6 (containing less active rAC). As can beseen in FIG. 1B, the preparation with less active enzyme (Lot 6)provided better results in IVF (fewer apoptotic embryos). This wasunexpected.

Lot 6 and Lot 7 were also tested using cultured rat chondrocytes (FIG.1C). At two weeks the amount of collagen 2 expression was analyzed usingwestern blotting. Cells cultured with Lot 7 (more active rAC) producedless collagen 2. Lot 6 was better in maintaining the chondrocytephenotype after expansion (FIG. 1C, based on the expression of collagen2). This was unexpected.

The improved performance of rAC containing less active AC ishypothesized to be due to the shorter half-life of the active enzyme incultured cells (conversely the longer half-life of the precursor).

In order to manipulate the ratio of inactive to active enzyme, pH wasadjusted to 4.0 and the preparation was incubated at 37° C. Under theseconditions an increase of approximately 10% active enzyme was observedfor every 3 hours of incubation. Thus, to covert a preparation that is90:10 inactive:active to 100% active, the preparation is incubated for27 hours.

An important variable here is temperature. If the preparations aremaintained (pH adjusted) frozen, there is no conversion. If thepreparations are maintained at 4° C. (in a refrigerator) the conversionproceeds but at 1% the efficiency of 37° C. (10% increase in activeenzyme requires 300 versus 3 hours). If the preparation is maintained atroom temperature (25° C.), it proceeds at 10% the efficiency (10%increase requires 30 hours). If the pH is not acidified, there is noconversion at 4° C. and only 1% conversion rate (300 hours are requiredfor an increase of 10%) at room temperature.

Example 3—Removal of Contaminating Acid Sphingomyelinase Activity fromrAC

Methods of removing contaminating acid sphingomyelinase activity (ASM)from the rAC preparations were developed. This requires incubation ofthe final rAC preparations at 60° C. for 10-20 minutes. This incubationdoes not affect rAC (activity or ratio of inactive to active) butremoves all ASM activity, which is essential to manufacturing (FIG. 2).

Example 4—Incubation of Media Containing Recombinant Human AcidCeramidase at 37° C. for Varying Lengths of Time

Conditioned media (DMEM, pH 6.8 containing 10% fetal calf serum) wascollected from Chinese hamster ovary cells overexpressing and secretingrecombinant human acid ceramidase (rhAC) (He et al., “Purification andCharacterization of Recombinant, Human Acid Ceramidase. CatalyticReactions and Interactions With Acid Sphingomyelinase,” J. Biol. Chem.278:32978-32986 (2003), which is hereby incorporated by reference in itsentirety). The cells were grown until ˜100% confluency in T-75 mmflasks, and media was then collected after 4 days of additional growth.The collected media was filtered through 0.22 mm membranes to removeddebris and placed in a 37° C. incubator for varying lengths of time. Atthe end of the incubation period the media was frozen at −20° C. priorto assay. AC activity (FIG. 3) was determined as previously described(He et al., Anal Biochem, 274:264 (1999), which is hereby incorporatedby reference in its entirety): reaction mixtures were incubated at 37°C. for one hour. AC Western Blot (FIG. 4): 6.5 μl/lane, was developedusing a mouse anti-human AC monoclonal antibody (1:300, #SC136275, SantaCruz) against the alpha-subunit. This data shows that in vitroincubation of media containing rhAC at 37° C. for 3-17 days, resultingin conversion of inactive precursor into active enzyme (represented bythe alpha subunit and an increase in enzymatic activity).

Example 5—In Vitro Conversion of Purified, Recombinant Human AcidCeramidase at 37° C.

Purified recombinant human AC (rhAC; 4 ug/ul in EMEM, pH 6.8) wasisolated from the media of overexpressing Chinese hamster ovary cells aspreviously described (He et al., “Purification and Characterization ofRecombinant, Human Acid Ceramidase. Catalytic Reactions and InteractionsWith Acid Sphingomyelinase,” J. Biol. Chem. 278:32978-32986 (2003),which is hereby incorporated by reference in its entirety). AC WesternBlot (FIG. 5): 6.5 μl/lane, was developed using a mouse anti-human ACmonoclonal antibody against the alpha-subunit (1:300, #SC136275, SantaCruz). This data shows that in vitro incubation of purified rhAC at 37°C. for 24 h (FIG. 5A) resulted in complete conversion of the precursorto active form. Incubation from 1-8 hours (FIG. 5B) showed a linearprogression of conversion.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed:
 1. A composition comprising a ceramidase mixturecomprising: an inactive acid ceramidase precursor; an active acidceramidase; and a pharmaceutically acceptable carrier, wherein thecomposition has no detectable acid sphingomyelinase activity.
 2. Thecomposition of claim 1, wherein the ceramidase mixture further comprisesa nucleic acid encoding the active acid ceramidase, a nucleic acidencoding the inactive ceramidase precursor, or a combination thereof. 3.The composition of claim 1, wherein the ceramidase mixture is in solidor liquid form.
 4. The composition of claim 3, wherein the ceramidasemixture is in a sterile injectable solution.
 5. The composition of claim3, wherein the ceramidase mixture is in a sterile dispersion.
 6. Thecomposition of claim 3, wherein the ceramidase mixture is in a formselected from tablets, capsules, elixirs, suspensions, a solution, adispersion, or syrups.
 7. The composition of claim 1, wherein theceramidase mixture further comprises one or more of the following: abinder, an excipient, a disintegrating agent, a lubricant, a sweeteningagent, or a liquid carrier.
 8. The composition of claim 1, wherein theceramidase mixture further comprises one or more of the following: asurfactant, glycerol, liquid polyethylene glycol, oil, saline, water,ethanol, a polyol, or a sugar solution.
 9. The composition of claim 8,wherein the ceramidase mixture further comprises saline or water. 10.The composition of claim 1, wherein the ceramidase mixture is in aerosolform.
 11. The composition of claim 10, wherein the aerosol form furthercomprises a propellant.
 12. The composition of claim 1, furthercomprising one or more additional agents that reduce ceramide levels.13. The composition of claim 12, wherein the one or more additionalagent that reduces ceramide levels comprise one or more inhibitors ofacid sphingomyelinase, inhibitors of ceramide synthases, or acombination thereof.
 14. The composition of claim 1, wherein the activeacid ceramidase and the inactive ceramidase precursor comprise the aminoacid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO. 5, SEQ ID NO: 6, or SEQ ID NO: 7.